1. Field of the Invention
The present invention relates to a biocatalytic process to produce optically active phenylalanine analogs from arylacrylic acids. In this process yeast containing L-phenylalanine ammonia lyase ("PAL") introduce a molecule of ammonia stereoselectively onto the double bond of a 3-substituted acrylic acid. The substituent at the 3 position of the 3-substituted acrylic acid is an aryl substituent including, for example, substituted phenyl groups, five member aromatic heterocyclics, and six member aromatic heterocyclics. In particular, this invention relates to a process for preparing phenylalanine-like aromatic amino acids using phenylalanine ammonia-lyase containing yeast, Rhodotorula graminis ATCC 20804.
2. Related Background Art
Phenylalanine and its derivatives have been used as essential building blocks in the construction of various types of biologically active molecules. An extremely important and powerful class of pharmaceutical compounds called protease inhibitors contain a phenylalanine-like architecture as their pharmacophores. These protease inhibitor drugs, especially HIV-protease inhibitors are appearing in increasing numbers in pharmacies and hospitals. The three recently approved protease-inhibitor drugs for treatment of AIDS all incorporate a phenylalanine derived moiety in their chemical structures. The search for more potent and effective protease inhibitors is continuing not only in the treatment of HIV infections but also in other disease area such as influenza and human cytomegalovirus (CMV). Currently, there is a pressing need for a general process of preparing a variety of optically active unnatural amino acids having phenylalanine-like structures as chiral synthons for synthesis of these drug candidates.
Although phenylalanine can be readily prepared in large scales by various existing methods including chemical and biological transformations, its analogs with various substitutions on the aromatic ring or with different aromatic ring systems such as naphthyl and heterocyclic ring systems, have been much more difficult to synthesize.
Trans-cinnamic acid ("t-cinnamic acid") and ammonia reversibly forms L-phenylalanine through the addition of the amino group ("amination") to the double bond of t-cinnamic acid. Elimination of ammonium ions from phenylalanine is called "deamination". The reaction is catalyzed by Phenylalanine Ammonia-Lyase (PAL) in either direction. Examples in the prior art of use and production of PAL, and of methods to produce phenylalanine with the assistance of PAL, include the references below.
U.S. Pat. No. 4,584,273, Finkelman et al., herein incorporated by reference in its entirety, discloses a method for the production of PAL by fermentation.
U.S. Pat. No. 4,598,047, McGuire, herein incorporated by reference in its entirety, discloses a method for producing catabolite resistant, PAL-producing mutant bacteria and yeast. The PAL thus produced is disclosed as being usable to make L-phenylalanine.
Hodgins, D., Journal of Biological Chemistry, 1971, 2977-2985, herein incorporated by reference in its entirety, reports on PAL purified from Rhodotorula glutinis, and the inhibiting effects of various ring-substituted analogues on phenylalanine deamination.
D'Cunha, G., Satyanarayan, V., and Nair, P., Enzyme and Microbial Technology, 1996, 421-427, herein incorporated by reference in its entirety, reports a procedure for the direct one-step synthesis of L-phenylalanine methyl ester by using PAL.
Rees, D., and Jones, D., Enzyme and Microbial Technology, 1996, 282-288, herein incorporated by reference in its entirety, reports producing PAL from Rhodosporidium toruloides. The stability of the PAL was also studied.
Evans, C., Choma, C., Peterson, W., and Misawa, M., Biotechnology and Bioengineering, 1996, 282-288, herein incorporated by reference in its entirety, reports the use of immobilized cells from a PAL-containing yeast mutant FP10M6 derived from rhodotorula rubra to demonstrate the feasibility of a continuous process of L-phenylalanine production from t-cinnamic acid.
Nakamichi, K., Nabe, K., Yamada, S., and Chibata, I., European Journal of Applied Biotechnology, 1983, 158-162, herein incorporated by reference in its entirety, reports the effect of various amino acids such as D-phenylalanine, D and L isoleucine, D and L leucine, L-valine, L-methionine, L-tryptophan, and L-tyrosine on the PAL activity in Rhodotorula glutinis.
U.S. Pat. No. 4,574,117, Vollmer et al., herein incorporated by reference in its entirety, discloses a method for producing L-phenylalanine from t-cinnamic acid and ammonia or ammonium salts using PAL as a catalyst. Reducing agents such as hydrogen sulfide, thioglycolic acid, thiosulfuric acid, nitrous acid, sulfurous acid, ammonium and metal salts of the above, dithiothreitol, ethylmercaptan, ethylenemercaptan, methylmercaptan, 2-mercaptoethanol, hydrogen, nitrous oxide, iron (II) compounds, manganese (II) compounds, sulfur, and zinc are added to the bioreaction mixture to reduce the effects of oxygen on catalyst life.
U.S. Pat. No. 4,584,269, Vollmer et al., herein incorporated by reference in its entirety, discloses a method for the PAL enzymatic conversion of t-cinnamic acid and ammonia to L-phenylalanine under substantially anaerobic, static conditions.
U.S. Pat. No. 4,562,151, Kishore, herein incorporated by reference in its entirety, discloses a method for producing L-phenylalanines and analogues from t-cinnamate and ammonia with the use of polyhydric alcohols to enhance instantaneous rates of reaction and to inhibit inactivation of the enzyme.
U.S. Pat. No. 4,757,015, Orndorff, herein incorporated by reference in its entirety, discloses a method for producing phenylalanine from t-cinnamate and ammonia with the use of Rhodotorula graminis ATCC 20804.
JP 06113870 A2, describes a procedure for the direct one-step enzymatic conversion of trans-cinnamyl methyl ester to L-phenylalanine methyl ester. The reverse reaction of phenylalanine ammonia lyase from Rhodotorula glutinis was utilized for this conversion.
JP 60043393 A, describes production of L-phenylalanine (LPA) by reacting cinnamic acid with ammonia or an ammonia-releasing substance in an aqueous medium in presence of a culture bath, cells or their processed material from a microorganism having L-phenylalanine ammonia lyase activity. The microorganism includes Rhodotorula rubra ATCC 20258, Rhodotorula texensis IFO 932, Rhodotorula glutinis IFO 0559, and Sporobolomyces IFO 1040.
JP 61043993 A, describes catalyzed formation of alpha-amino acids from 2-en-carboxylic acids and excess NH.sub.3 reacted in H.sub.2 O under anaerobic conditions in the presence of ammonia-lyase or mycelia such as Sporobolomyces roseus IF01040 containing ammonia-lyase or a substance derived from ammonia-lyase.
JP 06113870 A, describes the preparation of beta-substituted alanine derivatives of formula R--CH.sub.2 --CH(NH.sub.2)--CO.sub.2 H, comprising the reaction of ammonia with the acrylic acid derivative of formula R--CH.dbd.CHCO.sub.2 H in the presence of L-phenyl alanine ammonia-lyase. In the formulae, R is a monovalent group derived from a heterocyclic compound. Preferably, R=pyridyl, furyl or thienyl. Suitable enzymes include one or a mixture of the bacterial body obtained by culturing the transformant of Escherichia coli, MT10243 (FERM P-9023), integrated with the gene of the L-phenyl alanine ammonia-lyase originating from Rhodosporidium L-phenyl alanine ammonia-lyase producing bacterium (bacteria), treated products of the bacterial body, such as the washed, dried, crushed and fixed product, and purified enzyme extracted from the body or products.
A great volume of research is devoted to the application of PAL, as shown by the references above. Much work has been done with prokaryotic vectors to derive PAL catalysis of the cinnamic acid/phenylalanine reaction. Several eukaryotic yeast derived PAL have been used to catalyze the formation of phenylalanine. However, few yeast derived PAL have been used to produce a variety of optically active unnatural amino acids having phenylalanine-like structures as chiral synthons for synthesis. In particular, Rhodotorula graminis ATCC 20804 expressed PAL has not been used to introduce a molecule of ammonia stereospecifically into the double bond of the composition, ##STR1## to generate the optically active amino acid of the composition, ##STR2## wherein R is a substituted aromatic six-member ring system or a six or five-membered heterocyclic ring system including single and multiple substituted analogs.